Scientific Summary The incidence of Crohn's Disease (CD) is increasing, especially in young children and infants, posing an increasing burden on society.(1, 2) CD is believed to be the result of a failure of tolerance induction to normal intestinal bacteria in genetically predisposed individuals.(3) Tolerance is mediated by regulatory T cells (Tregs). Few treatments seek to augment the suppressive aspects of the immune system such as Tregs. Instead, current treatments rely on modifying the immune system by inhibiting pro-inflammatory responses. These therapies can have serious side effects, especially in children whose growth and development can be permanently affected. There is an urgent need for new therapies that have less potential side effects. Few treatments have sought to utilize Tregs via bystander suppression, and none have done so through the skin, as a therapy to suppress inflammation in the gut. CD patients have an inherent defect in the ability to form tolerance via the gut.(4, 5) Thus, attempts at inducing oral tolerance as a treatment for CD have so far not been successful,(6) and other routes to induce tolerance should be explored. Our studies have shown that the skin is a highly active immune organ capable of inducing Tregs and systemic immune tolerance. Importantly, epicutaneous exposure to the model antigen ovalbumin was able to abrogate and treat colitis and ileitis. However, the use of ovalbumin in human disease is impractical given that most people are already tolerant to ovalbumin. In contrast, flagellin may have excellent efficacy in patients who have aberrant immune responses to it already. Flagellin is the first antigen to be shown in both murine and human disease to have direct pathogenic effects in the development of colitis.(11, 12) CBir1 in particular is a dominant flagellin antigen with a role in both innate and adaptive immunity in mouse models of colitis.(12-16) We therefore propose to develop a novel, innovative approach to treat Crohn's disease by epicutaneously inducing Tregs to suppress intestinal inflammation using CBir1 flagellin epitopes. As the next step towards translating our findings into a therapy for treating Crohn's disease, we will perform studies determining the potential of epicutaneous tolerance induction using CBir1 flagellin epitopes. We will begin by determining the optimal dose of epicutaneous CBir1 flagellin epitopes needed to induce systemic tolerance and Tregs, and then determine if they function to suppress colitis. This work will be done in parallel with on-going work examining the mechanism of epicutaneous tolerance induction that is part of my K08 award. The central hypothesis of this application is that epicutaneous exposure to a novel antigen will generate tolerance and an induction of Tregs, which will control inflammation in CD directly and via bystander suppression. These studies utilizing Cbir1 flagellin epitopes are necessary pre-clinical studies before we can perform clinical trials. Data from these studies will justify transitioning to human studies.